13. (Optional, recommended) Perform antigen retrieval to unmask the antigenic epitope. The most commonly used antigen retrieval is a citrate buffer method. Arrange the slides in a staining container. Pour 300 ml of 10 mM citrate buffer, pH 6.0 into the staining container and incubate it at 95-100°C for 10 min (optimal incubation time should be determined by user). Remove the staining container to room temperature and allow the slides to cool for 20 min.

19. Apply 100 μl appropriately diluted biotinylated secondary antibody (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min.

20. Wash slides with PBS for 2 times, 5 min each.

21. Apply 100 μl appropriately diluted Sav-HRP conjugates (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min (keep protected from light).

22. Wash slides with PBS for 2 times, 5 min each.

23. Apply 100 μl DAB substrate solution (freshly made just before use: 0.05% DAB - 0.015% H2O2 in PBS) to the sections on the slides to reveal the color of antibody staining. Allow the color development for < 5 min until the desired color intensity is reached. (Caution: DAB is a suspect carcinogen. Handle with care. Wear gloves, lab coat and eye protection.)